These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.
Abstract
Prion diseases are neurodegenerative disorders associated with the structural conversion of the cellular prion protein (PrPc) into its misfolded infectious isoform (PrPSc). Despite substantial efforts, no disease-modifying therapy or cure is currently available. Here, we present an integrated computational-experimental pipeline for the rational design of cyclic peptides targeting PrPc to inhibit its pathogenic conversion. Starting from crystal structures of antibody-bound mouse PrPc, we develop a rational design strategy combined with iterative molecular dynamics simulations and sequence optimization to generate peptides with enhanced binding and structural impact. Three candidates were selected for experimental validation. Our results show that (49YGPDPSDSYT58, antibody numbering) that binds stably to the α2–α3 interface most effectively reduced PrPSc levels in GT1-7 cells, essentially by inducing allosteric re-arrangements that reinforce the intramolecular helical bundle. (89GQSNTKPYT97) and (89RQSNTWPYT97) binding the β1-α1/α3 junction exerted more modest effects due to the potential competition of the flexible tail to bind at this site. These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.
The pathogenic conversion of the cellular prion protein (PrPC) into the β-sheet-rich isoform PrPSc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive noncanonical amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substrate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substrate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and noninfectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new strategy for studying conformational dynamics in amyloid-forming proteins.
Jessica de Alcantara Ferreira, Daniel J. Walsh, Evelyn M. Turnbaugh et al.· Biochemistry· 0 citations
Prion diseases are neurodegenerative disorders characterized by the conformational conversion of the cellular prion protein (PrP
C
) into its pathogenic fibrillar form (PrP
Sc
). Recent cryo‐electron microscopy studies revealed that the mouse‐adapted RML prion fibril adopts an infectious single‐filament architecture with distinct N‐ and C‐terminal lobes, providing a structurally defined target for inhibitor design. Curcumin has been reported to interfere with amyloid aggregation through hydrophobic and π–π interactions; however, its structural determinants in prion fibril inhibition remain unclear. Here, curcumin‐like compounds were systematically explored to elucidate structure–activity relationships and identify potential inhibitors targeting the single‐filament RML prion fibril using structure‐based virtual screening, molecular docking, and molecular dynamics (MD) simulations. Five compounds were identified to preferentially bind to a hotspot region within the N‐terminal lobe. MD simulations revealed increased structural fluctuations upon ligand binding, as reflected by elevated root‐mean‐square deviation (RMSD) values (~20–25 Å), representing a structural relaxation toward a stable steady state while maintaining local dynamic features consistent with cryo‐EM data. Binding energy analysis indicated that van der Waals interactions dominate ligand–fibril association, consistent with ligand localization within a hydrophobic hotspot, suggesting a mechanism that modulates fibril dynamics rather than directly disrupting the fibril core. These findings suggest that curcumin‐like compounds modulate fibril stability through binding to a specific hydrophobic hotspot rather than directly disrupting β‐sheet structures, providing mechanistic insights into their potential as prion fibril inhibitors.
Muhammad Rifqi Nur Ramadani, Cheng-Ping Jheng, N. Jadid et al.· Journal of the Chinese Chemi...· 0 citations
This study enhances the understanding of the E. coli-genome nature and suggests the existence of specific and experimentally testable novel prions in this organism, and moves a step forward towards the identification of new prion proteins in bacteria.
Katherine Shreeve, Jinoh Jang, Mr. S Srivathsan et al.· Proteins: Structure, Functio...· 0 citations
The findings indicate that RNA aptamers, such as 1R6, may represent promising therapeutic candidates for synucleinopathies, thus opening new avenues in the treatment of these diseases.
K. Murakami, Thi Hong Van Nguyen, L. Tsuda et al.· bioRxiv· 1 citation
It is shown that stable knock-down of the protein disulfide isomerase P4HB in prion-susceptible CAD5 cells reduces PrPC levels and hinders the generation of protease-resistant PrP (PrPres) following infection with two different prion strains.
Genki Amano, H. Arshad, Zeel Patel et al.· PLoS Pathogens· 0 citations
The misfolding and aggregation of the prion protein into amyloid fibrils is primarily driven and stabilized by hydrophobic interactions, rendering the aggregates highly resistant to disaggregation and posing a significant therapeutic challenge. To address this issue, we design two compounds by covalently grafting isomeric ortho-vanillin or vanillin onto a MnMo6 cluster. The resulting o-Va-MnMo6 exhibits markedly superior disassembly activity, reducing neurotoxic PrP106-126 aggregates by 85% versus 49% for Va-MnMo6. The enhanced efficacy arises from the ortho-methoxy group of o-Va-MnMo6, which engages hydrophobic Ala113 via van der Waals interactions, whilst its hydroxyl group forms a hydrogen bond with the M112-A113 backbone. Concurrently, the polyoxometalate moiety electrostatically interacts with Lys110. The multipoint binding enables the molecule to straddle the contiguous K110HMA113 domain, effectively disrupting the aggregation core. In contrast, Va-MnMo6 interacts primarily with His111 via hydrogen bonding, lacking critical hydrophobic contact. Molecular dynamics simulations confirm that both compounds initially anchor electrostatically to Lys110, after which their differing ligands guide them to distinct sites, resulting in the divergent potencies.
